Possible role of sarcolemmal Ca2+/Mg2+ ATPase in heart function

The Ca2+/Mg2+ ATPase, which is activated by millimolar concentrations of Ca2+ or Mg2+, has been found to exist in the heart sarcolemmal membrane. Although a great deal of work has been carried out to characterize the enzyme activity as well as to purify the enzyme, the physiological function of the enzyme remains to be elucidated. This enzyme has been shown to be different from other known ion transport ATPases such as Na(+)-K+ ATPase and Ca2(+)-stimulated ATPase in terms of the activation kinetics of various cations, the sensitivity towards specific inhibitors, and the molecular weights. In view of the existing data and circumstantial evidence, it has been proposed that the Ca2+/Mg2+ ATPase is involved in the entry of Ca2+ into the cardiac cells, the transport of Mg2+ across the cell membrane, and extracellular ATP signal transduction.     

ATPase activity of purified plasma membranes and digestive vacuoles from Plasmodium falciparum

The ATPase activity of the human malaria parasite, Plasmodium falciparum was investigated using two experimental systems, (i) digestive vacuoles, and (ii) purified plasma membranes isolated from a chloroquine-sensitive and a chloroquine-resistant strain. No correlation between the level of ATPase activity and chloroquine sensitivity could be detected. In both systems, the ATPase activity of the chloroquine-resistant and -sensitive strain was decreased in the presence of the P-glycoprotein inhibitor vanadate. Susceptibility to inhibition by vanadate together with the lack of effect of ouabain implies a P-type ATPase activity in the plasma membrane. Furthermore, the inhibition of Fac8 ATPase activity by oligomycin both in the digestive vacuoles and the plasma membranes would be consistent with higher levels of Pgh1 in Fac8. Our data are consistent with the presence of a V-type H+-ATPase in the parasite food vacuole. Bafilomycin A1 and N-ethylmaleimide decreased the vacuolar ATPase activity in both chloroquine-resistant and -sensitive strains. Interestingly, a 30% decrease was observed between the ATPase activity of plasma membranes isolated from Fac8 and D10 in the presence of bafilomycin A1, suggesting the presence of a V-type ATPase in D10 plasma membrane that is underexpressed or altered in the plasma membrane of the chloroquine-resistant Fac8. The chemosensitisers tested had no effect on the ATPase activity of chloroquine-resistant P. falciparum in both systems suggesting that their activity is not mediated through an ATP-dependent mechanism. No effect was observed on the vacuolar ATPase activity in the presence of the antimalarials tested indicating that an ATP-dependent transport has not been activated.     

高血压大鼠红细胞膜钙泵功能障碍机理的初步分析

本工作比较自发性高血压大鼠(SHR)和肾性高血压大鼠(RHR)及对照大鼠红细胞膜Ca~(2+),Mg~(2+)-ATP酶(钙泵)活性及其对CaM、TFP、川芎嗪和硝苯吡啶(Nifedipine、Nif)等的反应,目的是分析高血压时钙泵功能障碍的机理,并寻找能有效提高钙泵活性的药物,为高血压防治提供有效措施。 结果表明SHR及RHR基础钙泵活性明显低于相应对照大鼠钙泵活性;原因之一可能与高血压动物质膜钙泵对钙泵抑制剂TFP的敏感性增加有关,对RHR,川芎嗪作用很类似TFP。提示此药有CaM拮抗作用。Nif除了公认为钙通道阻断剂外,本实验表明对RHR它还有激活钙泵的作用。     

Inhibitory effect of insulin and cytoplasmic factor(s) on brain (Na(+) + K+) ATPase.

(Na+ + K+)ATPase activity in cerebral cortex was modulated by insulin action depending on the Mg2+ concentration. Thus, in homogenates in the presence of 1-3 mM Mg2+, insulin stimulated the enzyme, whereas in the presence of 4-6 mM Mg2+ inhibition was observed. Exposure of synaptosomal membranes to the soluble fraction resulted in inhibition of ATPase activity in a dose-dependent manner. The inhibitory effect of insulin was regulated by a cytoplasmic factor in a dose-dependent manner. Similar variations to those obtained with a crude synaptosomal fraction were obtained by using a partially purified ATPase. These results indicated the importance of soluble factors in the modulation of ATPase by insulin and add more evidence in support for a role of insulin as a neuromodulator.     

Cryptic adenosine triphosphatase activities in plasma membranes of CCl4-cirrhotic rats. Its modulation by changes in cholesterol/phospholipid ratios

The activities of Na+,K+-, and Ca2+-ATPases were determined in plasma membranes obtained from livers of rats treated acutely and chronically with CCl4. Twenty-four hours after a single oral dose of CCl4 the ATPases decreased below 50% of control values. The activity of Ca2+-ATPase returned to normal after 4 days, and Na+,K+-ATPase activity returned to normal values after 12 days. One week after initiation of the chronic intraperitoneal treatment with CCl4, the Na,K+-ATPase decreased to 40% of control values and continued to decrease further until reaching values below 1%. Ca2+-ATPase followed a pattern similar to that obtained with Na+,K+-ATPase, except that the decrease was not as severe. Colchicine treatment prevented the modifications in ATPases when given simultaneously with CCl4 and reverted the alterations in ATPase activities of the CCl4-cirrhotic animals. Because ATPases are known to be modulated by the lipid composition of the membrane, we also determined the cholesterol to phospholipid ratio in all the isolated membranes. The ratios were increased in membranes with low ATPase activity due to an increase in the total concentration of cholesterol. Plasma membranes of cirrhotic rats treated with colchicine showed a low concentration of cholesterol, a decreased cholesterol to phospholipid ratio, and Na+,K+-ATPase activity was almost normal. When plasma membranes of cirrhotic rats were fused with phosphatidyl serine-containing liposomes, the cholesterol to phospholipid ratio decreased and the ATPase activity increased. The ATPase activity of normal plasma membranes decreased below 20% of control values when enriched with cholesterol. Our results suggest that the decrease in the plasma membrane Na+,K+-ATPase activity of the cirrhotic rat is due in part to an increase in its cholesterol concentration and in the cholesterol to phospholipid ratio.     

脑梗塞患者红细胞膜泵活性与血液流变性的相关性研究

目的 :探讨脑梗塞 (CI)病人红细胞膜Na+ K+ ATPase、Ca2 + Mg2 + ATPase与血液流变学指标的相关性。方法 :对 49例非急性期的脑梗塞患者和 2 9例健康人分别测定Na+ K+ ATPase、Ca2 + Mg2 +ATPase、Ox LDL、LDL C、Cho C以及血液流变学指标。结果 :CI组全血粘度、血浆粘度、还原粘度、HCT、EAI、TK与Na+ K+ ATPase、Ca2 + Mg2 + ATPase活力的变化呈明显负相关 ;与Ox LDL、LDL C、Cho C呈明显正相关。结论 :CI患者红细胞膜泵活性的变化能引起血液粘滞性的改变 ,其作用主要是红细胞膜Na+ K+ 泵、Ca2 + 泵的活性降低 ,导致红细胞变形能力下降。     

A high affinity Ca2+-dependent ATPase in the surface membrane of the bloodstream stage of Trypanosoma rhodesiense

Addition of Ca2+ (0.01-1 mM) to a standard Trypanosoma rhodesiense Mg2+-ATPase assay failed to elicit any increase in activity. However, in the absence of externally added Mg2+ and using calcium-EGTA or calcium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific Ca2+-ATPase. Cell fractionation studies revealed this ATPase to be predominantly associated with subcellular particles having an equilibrium density of 1.22 g cm-3 and identified as surface membrane. Using a discontinuous sucrose gradient, a surface membrane enriched (SME) fraction, only slightly contaminated with mitochondria as judged by dichlorophenolindophenol-linked alpha-glycerophosphate dehydrogenase activity, was prepared. The SME fraction exhibited Ca2+-ATPase activity, using 200 nM free Ca2+, of 90 and 21 mU mg-1 protein, respectively, using CDTA and EGTA as buffering ligands. This latter result was most unexpected and indicated that the Ca2+-ATPase, in addition to having no Mg2+ requirement, was inhibited by submicromolar levels of Mg2+. The Ca2+-ATPase was found to have a K0.5 = 128 +/- 22 nM free Ca2+, the response to increasing Ca2+ concentration displaying an extremely high degree of co-operativity (Hill number (nH) = 4.9). The enzyme was found to be highly substrate-specific for ATP with K0.5 = 6.2 +/- 0.61 microM ATP. A Hill plot of the reaction velocity as a function of ATP concentration indicated two substrate binding sites (nH = 1.55). A range of potential modulators of ATPase activity were investigated, with only vanadate (V2O3-8) having any effect: 47% inhibition at 5.0 microM. The Ca2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (50 microM), whilst addition of calmodulin failed to produce any stimulation of activity. It is concluded that the kinetic properties of this ATPase are compatible with a potential role in the regulation of intracellular Ca2+ in bloodstream T. rhodesiense.     

ATPase activity of erythrocyte membrane in patients with trisomy 21 (Down''s syndrome)

ATPase activity of crythroyte membranes was determined in 25 cases of Down's syndrome verified by cytological and psychological examinations. The age range of the patients was 8-25 years; 16 males and 9 females. Thirty health male volunteers were selected as the control group. There was a marked reduction of total ATPase, Na+, K+-ATPase, Mg++ATPase activities and rate of ouabain inhibition in the patients with Down's syndrome. The authors suggest that there might exist transport defects in the red cell membranes in such patients.     

Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway

Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number.     

Plasmodium falciparum-infected erythrocytes contain an adenylate cyclase with properties which differ from those of the host enzyme

It has been postulated that differentiation of the human malaria parasite, Plasmodium falciparum, is controlled by cAMP levels. We have determined that P. falciparum synthesizes an adenylate cyclase with several properties distinct from those of the mammalian host cell enzyme. Adenylate cyclase activity was compared in P. falciparum-infected erythrocytes, isolated parasites free of host cell material, and uninfected erythrocyte membranes. The parasite enzyme was unaffected by GTP gamma S, AlF4-, and forskolin, while the erythrocyte enzyme was markedly stimulated by each of these compounds. The parasite adenylate cyclase also exhibited a striking preference for Mn2+ over Mg2+, which was not evident in the erythrocyte enzyme. Moreover, differing cation and pH sensitivities were observed for adenylate cyclase activity in the two cell types. When infected and uninfected erythrocytes were compared, the basal adenylate cyclase activity of infected cells was 7 and 49 times that measured in uninfected erythrocytes in the presence of Mg2+ and Mn2+, respectively. Furthermore, adenylate cyclase activity in infected cells exhibited properties typical of the parasite enzyme. This indicates that synthesis of the parasite enzyme rather than stimulation of the host enzyme accounts for the increased activity in infected cells.     

高压氧对脑损伤家兔线粒体膜酶活性影响的实验研究

目的：研究外伤性脑水肿后线粒体Na^ ,K^ -ATP酶,Ca^2 ,Mg^2 -ATP 酶,Mg^2 -ATP酶活性,MDA含量在继发性脑损伤中的作用,以及高压氧治疗的机理。方法：应用家兔脑损伤模型进行高压氧治疗,观察伤后不同时间脑含水量,线粒体ATP酶活性、MDA含量变化,以及高压氧治疗对它们的影响。结果：线粒体三种ATP酶活性在伤后4h即开始下降,至48h降到最低点,MDA含量伤后显著增加,随时间延长增高更加明显,MDA与脑含水量间呈正相关,ATP酶活性与脑含水量之间呈负相关;高压氧组与对照组比较,上述指标变化均较轻。结论：脑外伤后线粒体自由基反应增强,ATP酶活性受抑,促进脑水肿发生、发展,高压氧通过减少自由基、增强ATP酶活性,减轻脑水肿。     

ATP-sensitive K+ transport in adrenal chromaffin granules

In the present study the influx of 86Rb+, a K+ analogue, was studied in mitochondria, microsomes and chromaffin granules prepared from adrenal gland medulla. The most active electrogenic 86Rb+ transport was found in the membrane fraction identified as chromaffin granules by marker enzyme estimation. The transport was found to be sensitive to ATP, ATP gamma S, ADP and to the triazine dyes, but not to AMP and cAMP. The inhibition induced by ATP was observed in the absence of externally added Mg2+, suggesting that a free nucleotide, rather than the ATP-Mg complex, was required for inhibition. Furthermore, the 86Rb+ influx was found to be inhibited by Mg2+ alone, but not by Ca2+ and antidiabetic sulfonylureas. The 86Rb+ influx was not stimulated by potassium channel openers. In conclusion, our results indicate that an electrogenic, ATP-sensitive potassium transport system operates in the chromaffin granule membrane.     

Properties of the HCO 3 − -stimulated Mg2+-ATPase activity in red cell membranes

The HCO-3-stimulated Mg2+ -ATPase activity in red cell ghost fragments was investigated. Increasing the HCO-3 concentration in the incubation medium resulted in increased ATPase activity. NaHCO3 appeared to be more effective than KHCO3 in this regard. The ATPase activities were slightly stimulated by increases in ionic strength and utilized ITP almost as readily as ATP. A Mg/ATP ratio of 1.0 and a pH of 7.6 yielded maximum activity. These properties are of interest since the present enzyme is the only unquestionable instance where a HCO-3 ATPase is located in the surface membrane of a cell.     

Some properties of the adenosine triphosphatase associated with herpes simplex virus and nuclear membrane of host cells.

An enzyme capable to split adenosine triphosphate (ATP) was shown to be firmly associated with mature herpes simplex virus particles purified from infected rabbit lung (ZP) cells. The enzyme localized in the viral envelope was markedly activated by bivalent cations, to the largest degree by Mg2+ at a pH optimum of 7.8--8.0. Na+ and K+ ions neither separately nor together showed any activating effect. Enzyme activity was not sensitive to the action of ouabain. No adenosine diphosphatase (ADPase) and adenosine monophosphatase (AMPase) activities were observed. ATPase activity was competitively inhibited by ADP. AMP and inorganic phosphate were without effect. The ATPase of nuclear membranes isolated from ZP cells exhibited similar properties but behaved differently to the action of sodium dithionite, dinitrophenol, oligomycin and gramicidin, as well as on heat inactivation. The origin of the virus enzyme is discussed.     

A circulating inhibitor of the platelet Na+,K+ adenosine triphosphatase (ATPase) enzyme in allergy.

Previous investigations have documented a reduced activity of the sodium-potassium-stimulated adenosine triphosphatase enzyme (Na+,K+ ATPase) in platelet membranes of allergic subjects. The purpose of this study was to determine if the reduced Na+,K+ ATPase activity was due to an enzyme inhibitor. Na+,K+ ATPase activity of a particulate fraction of sonicated platelets was determined by spectrophotometry in asymptomatic adults with and without allergy. The Na+,K+ ATPase level (mean, nanomoles per microgram of protein per minute; +/- STD) of allergic subjects (0.9 +/- 1.3) was lower (p less than 0.001) than that of nonallergic subjects (3.9 +/- 1.6). In contrast, when the same platelet fractions were frozen before assay, Na+,K+ ATPase was higher (p less than 0.005) in allergic subjects (6.0 +/- 1.4) than in nonallergic subjects (3.6 +/- 2.0). An inhibitor of canine kidney Na+,K+ ATPase was detected in the buffer in which these platelet fractions were frozen, allergic subjects (0.5% +/- 0.4% inhibition per microgram of protein) compared to nonallergic subjects (0.04% +/- 0.08%; p less than 0.005). The level of inhibition correlated positively with the postfreezing increase in platelet membrane Na+,K+ ATPase, suggesting a freezing-induced displacement of an inhibitor from the membrane. Plasma from these same subjects inhibited Na+,K+ ATPase activity of normal platelets, allergic subjects (70% +/- 31% inhibition) compared to nonallergic subjects (13% +/- 16%; p less than 0.001). These data suggest that the transport-enzyme defect observed in platelets from allergic subjects was due to a circulating Na+,K+ ATPase inhibitor. In vivo Na+,K+ ATPase inhibition in allergy could have profound effects on intracellular cation concentrations and broad implications for pathogenesis.     

Phenomenon of hot-cold hemolysis: chelator-induced lysis of sphingomyelinase-treated erythrocytes.

Staphylococcus aureus produces a phospholipase C specific for sphingomyelin (beta-hemolysin). Erythrocytes with approximately 50% sphingomyelin in their membranes, e.g., from sheep, have been shown to have up to 60% of this phospholipid hydrolyzed by this enzyme at 37 C in isotonic buffered saline without hemolysis. Cooling of sphingomyelinase C-treated erythrocytes to 4 C causes complete lysis of the cells, a phenomenon known as hot-cold hemolysis. The addition of ethylenediaminetetraacetate (EDTA) to sheep erythrocytes preincubated with sphingomyelinase C was found to induce rapid hemolysis at 37 C. The treated cells became susceptible to chelator-induced hemolysis and to hot-cold hemolysis simultaneously, and the degree of lysis of both mechanisms increased equally with prolonged preincubation with sphingomyelinase C. Erythrocytes of species not readily susceptible to hot-cold hemolysis were equally insusceptible to chelator-induced lysis. Chelators of the EDTA series were the most effective, whereas chelators more specific for Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+ were without effect. The rate of chelator-induced lysis was dependent on the preincubation period with beta-hemolysin and on the concentration of chelator added. The optimal concentration of EDTA was found to equal the amount of exogenously added Mg2+, a cation necessary for sphingomyelinase C activity. Hypotonicity increased the rate of chelator-induced hemolysis, whereas increasing the osmotic pressure to twice isotonic completely inhibited chelator-induced lysis. The data suggest that exogenously added and/or membrane-bound divalent cations are important for the stability of sphingomyelin-depleted membranes. The phenomenon of hot-cold hemolysis may be a consequence of the temperature dependence of divalent ion stabilization.     

Kinetics and pattern of organelle exocytosis duringToxoplasma gondii/host-cell interaction

The fate ofToxoplasma gondii dense-granule (GRA2, GRA3), rhoptry (ROP1), and surface (SAG1) proteins was followed by immunofluorescence assay (IFA) and immunoelectron microscopy at different stages after infection. Dense-granule exocytosis occurred in the apical area of the tachyzoite within minutes of invasion. Several exocytic events were found simultaneously in the same organism, both by serial sectioning and by freeze-fracture studies. Dense-granule contents were first found as a dense material trapped between parasite and vacuole membranes before either the vacuolar network or the vacuole membrane could be immunolabeled with specific antibodies. The vacuolar network was strongly labeled with dense-granule antibodies but not with the SAG1-specific probe, which suggests that the network is not enriched in membrane proteins. In addition to strongly labeling the vacuole membrane, GRA3 antibodies also labeled strands extending from the parasitophorous vacuoles into the host-cell cytoplasm.     

Liver plasma membrane enzyme activities following glutaraldehyde fixation.

The Wachstein-Meisel ATPase histochemical method has been previously used to demonstrate the ultrastructural localization of this enzyme in both whole liver and isolated plasma membranes following fixation in glutaraldehyde. In the present study biochemical assay, of liver plasma membrane enzymes following fixation in cold 2.5% glutaraldehyde showed that approximately 40% of Mg2+-ATPase, but only 4% of (Na+-K+)-ATPase activity remained in membranes from either control or ANIT-treated rats. In addition, 5'-nucleotidase activity was almost abolished by fixation. The present results indicate that the Wachstein-Meisel method, when applied to biliary canaliculi, can reliably be used to demonstrate the ultrastructural, histochemical localization of Mg2+-ATPase but not that of (NA+-K+)-ATPase. Furthermore, the method permits a valid comparison to be made of the relative Mg2+-ATPase activity in normal and chemically damaged biliary canaliculi.     

Effect of insulin upon membrane-bound (Na+ + K+)-ATPase extracted from frog skeletal muscle.

1. Insulin stimulates the activity of membrane-bound ATPase isolated from frog skeletal muscle and from rat brain. The increase in activity of the membrane-bound ATPase system isolated from frog ranged from 9-8 to 53% at concentrations of Na+ (25 mM), K+ (10 mM), and ATP (2 mM) similar to those in in vivo experiments conducted previously (Moore, 1973). The increased activity of the membrane-bound ATPase is, therefore, at least as great as the insulin-induced increase in Na efflux (10-38%) from intact cells (Moore, 1973). If the concentration of Na+ is lowered to 4 mM and that of ATP lowered to 0-5 mM albumin, and 10(6) M, the increase in ouabain-inhibitable ATPase activity can reach as high as 400%. 2. Ouabain, at a concentration (10(-3) M) sufficient to inhibit stimulation of the frog ATPase by increasing Na from 4 to 25 mM, completely blocked the stimulation of ATPase activity due to insulin. 3. At 2 mM-ATP, 100 mM-Na+, and 20 mM-K+, conditions which maximally activate the (Na+ + K+)-ATPase, insulin did not increase the ATPase, activity. Stimulation was consistently seen at 10 mM-K+, 0-5 mM-ATP, and either 4 mM or 25 mM-Na+. 4. The finding that insulin does not stimulate the ATPase activity in conditions in which the (Na+ + K+)-ATPase component is maximally activated and especially the fact that ouabain can reproducibly inhibit insulin stimulation of the membrane-bound ATPase activity strongly suggest that interaction of insulin with its receptor upon the plasma membrane somehow stimulates the (Na+ + K+)-ATPase system (ouabain sensitive; ATP phosphohydrolase, EC (3.6.1.3). These results are consistent with previous studies of the effect of insulin upon Na efflux from intact cells (Moore, 1973) and support the previous conclusion that the component of Na efflux stimulated by insulin is active. The evidence suggests that insulin probably does not affect Vmax of the (Na+ + K+)-ATPase system, but may increase the affinity of the enzyme system to one or more effectors, most likely Na+, ATP, and perhaps K+. 5. Oxidized glutathione (2-7 X 10(-6) M), 10(-6) M, 10(-7) M, and 10(-8) M cyclic AMP did not affect the ATPase activity 10(-6)Malbumin, and . 6. The results are consistent with the view that the Na pump, (Na+ + K+)-ATPase, is intimately involved with the physiological action of insulin and may be transducer between the binding of insulin to its receptor on the plasma membrane and the cellular actions of insulin.     

Degradation of gonococcal outer membrane proteins by human neutrophil lysosomal proteases.

Interest in the molecular mechanisms of leukocyte bactericidal activity led us to study the effects of human neutrophil lysosomal proteases on the outer membrane (OM) proteins of Neisseria gonorrhoeae. A protease fraction containing cathepsin G and elastase activity was partially purified by gel filtration chromatography of acetate extracts of purified neutrophil granules. OM was obtained from gonococci by French press-Sarkosyl or by LiCl2 extraction. The principal (protein I) and opacity-associated (proteins II) OM proteins of N. gonorrhoeae were hydrolyzed by lysosomal proteases; proteins II were more susceptible to hydrolysis than protein I. Treatment of whole gonococci, with subsequent purification of OM, or direct treatment of purified OM led to identical hydrolysis of OM proteins by lysosomal proteases as indicated by sodium dodecyl sulfate-polyacrylamide gel patterns. Similarly, hydrolysis of purified OM proteins was identical whether OM was treated with unfractionated granule extract or with the partially purified lysosomal proteases, indicating that the observed hydrolysis by unfractionated lysosomal contents was due solely to the lysosomal protease fraction. Hydrolysis of OM proteins was dependent upon the concentration of proteases, time, and temperature. Hydrolysis of proteins II was observed with as little as 1 microgram of proteases per ml for 1 h at 37 degrees C. OM incubated alone or with heat-inactivated proteases showed no hydrolytic activity. The addition of 25 mM Na+, K+, Mg2+, or Ca2+ to incubation mixtures containing proteases and OM did not alter hydrolytic activity as indicated by sodium dodecyl sulfate-polyacrylamide gel patterns.